A ubiquitin-based tagging system for controlled modulation of protein stability

Many biotechnology applications depend on the expression of exogenous proteins in a predictable and controllable manner. A key determinant of the intracellular concentration of a given protein is its stability or "half-life." We have developed a versatile and reliable system for producing short half-life forms of proteins expressed in mammalian cells. The system consists of a series of destabilization domains composed of varying numbers of a mutant form of ubiquitin (Ub(G76V)) that cannot be cleaved by ubiquitin hydrolases. We show that increasing the number of Ub(G76V) moieties within the destabilization domain results in a graded decrease in protein half-life and steady-state levels when fused to heterologous reporter proteins as well as cellular proteins. Cells expressing a destabilized P-lactamase reporter act as a robust, high-throughput screening (HTS)-compatible assay for proteasome activity within cells.